This project aims to elucidate how the architecture of the transitional ER (tER) and Golgi apparatus are linked to the actions of individual molecular components. The experimental system is two budding yeasts: Pichia pastoris, which has discrete tER sites adjacent to Golgi stacks, and Saccharomyces cerevisiae, which has a delocalized tER and a fragmented Golgi. Initial results indicate that Sec16p is a key determinant of Ter organization, and that Golgi cisternae in P. pastoris are attached to each other and to tER sites by a ribosome-excluding "matrix" that includes the protein Grh1p. The working hypothesis to be tested is that tER proteins and Golgi matrix proteins have conserved functions in membrane traffic, but also have variable features that influence the architecture of the tER-Golgi system. Specific Aim #1 is to determine how Sec16p regulates tER organization. Experiments will test the model that P. pastoris Sec16p self-associates and thereby crosslinks patches of COPII coat proteins to make tER sites, whereas S. cerevisiae Sec16p does not self-associate and therefore does not make tER sites. Specific Aim #2 is to elucidate the roles of Grh1p and other cis-Golgi matrix proteins in organizing the tER-Golgi system. Grh1p may interact with a partner protein to hold Golgi stacks next to tER sites. This hypothesis will be tested by in vivo and in vitro analyses of Grh1p and other matrix components. Specific Aim #3 is to initiate an in vivo reconstitution of tER sites and Golgi stacks in S. cerevisiae. The ultimate goal is to identify the full set of proteins responsible for organizing tER-Golgi in P. pastoris, and then transfer the corresponding genes to S. cerevisiae. Abnormal secretory pathway function is a causative agent in disorders such as cancer and autoimmune diseases. Adequate treatments will require a cell biological understanding of the processes that define secretory compartments. The present work aims to reveal these basic principles of cellular organization.